Figure 5.

Wnt/β-catenin signaling transcriptionally represses miR-139-5p in KRAS-mutant CRC cells. (A) Left, immunoblots of β-catenin expression in DKs-8 and Caco-2 cells transfected with β-catenin and control plasmids. Right, miR-139-5p expression detected in the indicated cells by qRT-PCR. (B) Left, immunoblots of β-catenin expression in DKO-1 and SW620 cells transfected with β-catenin siRNA (siβ-catenin) or control siRNA (siCtrl). Right, miR-139-5p expression detected in the indicated cells by qRT-PCR. (C) DKs-8 and Caco-2 cells were treated with Wnt3a (5 ng/mL) or DMSO for 24 h. miR-139-5p expression was detected by qRT-PCR. (D) DKO-1 and SW620 cells were treated with ICG-001 (2 µg/mL) or DMSO for 24 h. miR-139-5p expression was detected by qRT-PCR. (E) DKs-8 cells were transfected with TCF3, TCF4, or control plasmid for 48 h. Immunoblotting was performed to examine the expression of TCF3 and TCF4. miR-139-5p expression was detected by qRT-PCR. (F) DKO-1 cells were transfected with TCF3 siRNA (siTCF3) and TCF4 siRNA (siTCF4) for 48 h. Immunoblotting was performed to examine the expression of TCF3 and TCF4. miR-139-5p expression was detected by qRT-PCR. (G) Left, schematic representation of consecutive deletion constructs spanning the promoter region (-5000 to +1) of MIR139, with the site -2373 nucleotides upstream of MIR139 sequence as the transcription start site ²³. The putative TCF4-binding sites in the miR-139 promoter region are shown in black boxes. Right, luciferase activity in DKO-1 cells transfected with the luciferase vector pGL3 driven by either the WT or deletion promoter. (H) Left, schematic representation of the mutation constructs of the miR-139 promoter. Right, luciferase activity in DKO-1 cells transfected with the luciferase vector pGL3 driven by either the WT or mutant promoter. (I) The ChIP assay demonstrated the direct binding of TCF4 to the miR-139 promoter in DKO-1 cells. qRT-PCR was carried out to quantitate the immunoprecipitated products using primers within the miR-139 promoter. Primers within the cyclin D1 and 16q22 promoters served as the positive and negative controls, respectively. *P< 0.05; **P <0.01; n.s., not significant.